Microfluidic devices are generally known, including immunoassay devices. Easy-to-use immunoassay devices are generally desired for point-of-care applications. For instance, “one-step” (immuno)assays akin the well known pregnancy test have been developed, wherein all reagents needed to detect an analyte of interest in a sample are integrated into the device during manufacturing. A non-expert user only needs to add a sample in a sample-receiving structure of the device. From there, the sample flows and redissolves the reagents, which then react with the analyte so as to make it detectable by means of, for example, optical or electrochemical methods. Typically, detection of analytes that have been reacted with the reagents occurs in a region of the device that is located after the area containing the reagents.
Point-of-care diagnostics should benefit from miniaturization based on microfluidics because microfluidics integrate functions that can together preserve valuable samples and reagents, increase sensitivity of a test, and accelerate mass transport limited reactions. Detection of several analytes in parallel is also facilitated by miniaturization. Finally, miniaturization increases the portability of diagnostics by reducing their size and weight. However, a challenge is to incorporate reagents into microfluidics and to make the devices simple to use. Yet another challenge is to make devices for one-step assays disposable and cheap to manufacture. For this reason, devices for one-step assays rarely have actuation mechanisms that interact with flow of liquids and reagents.
L. Gervais and E. Delamarche have recently demonstrated a concept for one-step immunoassays using microfluidic chips (Lab Chip, 2009, 9, 3330-3337). More in detail, the authors have integrated reagents such as detection antibodies (dAbs) and capture antibodies (cAbs) with microfluidic functional elements for detecting analyte molecules inside a sample using a one-step immunoassay: the integrated device only requires the addition of sample to trigger a cascade of events powered by capillary forces for effecting a sandwich immunoassay that is read using a fluorescence microscope. The microfluidic elements comprise a sample collector, delay valves, flow resistors, a deposition zone for dAbs, a reaction chamber sealed with a polydimethylsiloxane (PDMS) substrate, and a capillary pump and vents. Parameters for depositing 3.6 nL of a solution of dAb on the chip using an inkjet are optimized. This deposition by means of an inkjet is sometimes termed “spotting”. The PDMS substrate is patterned with receptors for analytes, which provide signal areas as well as positive control areas. Various storage conditions of the patterned PDMS were investigated for up to 6 months, revealing that storage with a desiccant preserved at least 51% of the activity of the cAbs. C-reactive protein (CRP), a general inflammation and cardiac marker, was detected with this one-step chip using only 5 μL of human serum, by measuring fluorescent signals from 30×100 μm2 areas of the PDMS substrate in the wet reaction chamber. In this example, the one-step chip can detect CRP at a concentration of 10 ng mL−1 in less than 3 min and below 1 ng mL−1 within 14 min.
In the above paper, three types of structures for spotting and dissolving the dAbs are disclosed, which are reproduced here on FIG. 1A-C. Namely:
FIG. 1A depicts a structure with main straight channel 21;
FIG. 1B shows a structure with a main channel 21, side connector 216 and spotting target 215; and
FIG. 1C shows a structure with a main channel 21, a plurality of side connectors 216, 217 and spotting target 215.
The geometries of said structures were developed with the sake of miniaturization (for better assay performances) while bringing spotted dAbs directly on the sample flow path or near to it so that dAbs can dissolve in the sample flowing through the device. Spotting small volumes of liquids in such devices (i.e., a few nL or tens of nL) is challenging but can be achieved thanks to inkjet technology.
More in detail, in FIG. 1A, it is difficult to position an inkjet over the main straight channel and the volume of dAb solution spotted must be kept small to prevent overflow or spreading of the spotted solution throughout the flow path defined therein. The structure of FIG. 1B and specially that of FIG. 1C helps relaxing the accuracy needed for spotting, thanks to the larger circular target area 215 on top of the side connector 216. Adding branching channels 217 furthermore gives the possibility to accommodate more spotted liquid, which might be needed to increase the amount of dAb available for a given test (e.g. high analyte concentration).
Next, FIG. 1D is a negative, grayscaled fluorescence microscope image showing the dAbs 200 in the deposition zone 215 (of FIG. 1B) after spotting and drying. Although the dAbs are clearly located inside the microstructures, an air bubble may form as soon as the sample starts entering the deposition zone. Moreover, most of the spotted dAbs may not diffuse/flow outside the deposition zone in that case.
In fact, the sensitivity of an assay is strongly affected by the time during which dAbs bind to analytes and surface-immobilized receptors and the volume of sample in which dAbs dissolve. There is a time/volume equivalence due to the flow rate equation: t=V/Q, where t is the time needed to displace a given volume V at a flow rate Q.
In a one-step assay, it might be realized that the dissolution profile of dAbs is hard to optimize because, ideally, no actuation on Q, the flow rate, is performed by an operator or instrument. Actuation of Q on a chip is possible but requires expensive and complex devices and a source of power for actuation.
To summarize, a structure with a main straight channel section as in FIG. 1A makes it challenging for spotting, owing to the x, y-in plane accuracy required, and a limited volume can be spotted. Moreover, it typically leads to a (too) fast dissolution of the spotted dAbs (typically 30 seconds), which in turn leads to a small volume only of sample containing the dAbs. In a microfluidic device (or miniaturized system), it might be realized that a liquid can dissolve reagents that are placed on a liquid path too efficiently. If reagents are too quickly dissolved, they may not be present in the correct volume fraction of liquid for a given test. For instance, dAbs that are too quickly dissolved will be present mostly at the filling front of a sample. These dAbs will not be able to react with many analytes in the sample and will quickly move over receptor areas without giving sufficient time for the receptors to gradually bind analytes and dAbs. Therefore, the efficiency of microfluidic devices in moving liquids and dissolving reagents can be (paradoxically) counterproductive.
Some examples of how to slow the release of reagents in microfluidic devices are provided below together with explanations on their limitations. In a structure such as depicted in FIG. 1B (main channel with side connector and spotting target), the sample flowing (from left to right) enters only partially the side connector, leading to an air bubble entrapment, limited dissolution of dAbs and poor diffusion of dAbs from the side channel to main channel on the left side. Adding a plurality of side connectors (FIG. 1C) can accommodate more dAb solution but essentially the same problem as in FIG. 1B remains. In addition, here, the absence of direct convection of liquid through the areas containing the dAbs makes the diffusion of the dAbs variable because the diffusion of the dAbs is strongly dependent on the coordinates of the dAbs in the structures after they have been deposited and dried.
Therefore, the prior art structures of FIG. 1 have many drawbacks. Most importantly, one may realize that such structures do not allow for an easy and precise control of the dissolution profile of dAbs. There is accordingly a need for an improved microfluidic device.